The cause of cystic fibrosis–related diabetes (CFRD) remains unknown, but cystic fibrosis transmembrane conductance regulator (CFTR) mutations contribute directly to multiple aspects of the cystic fibrosis phenotype. We hypothesized that susceptibility to islet dysfunction in cystic fibrosis is determined by the lack of functional CFTR. To address this, glycemia was assessed in CFTR null (CFTR−/−), C57BL/6J, and FVB/NJ mice after streptozotocin (STZ)-induced β-cell injury. Fasting blood glucose levels were similar among age-matched non–STZ-administered animals, but they were significantly higher in CFTR−/− mice 4 weeks after STZ administration (288.4 ± 97.4, 168.4 ± 35.9, and 188.0 ± 42.3 mg/dl for CFTR−/−, C57BL/6J, and FVB/NJ, respectively; P < 0.05). After intraperitoneal glucose administration, elevated blood glucose levels were also observed in STZ-administered CFTR−/− mice. STZ reduced islets among all strains; however, only CFTR−/− mice demonstrated a negative correlation between islet number and fasting blood glucose (P = 0.02). To determine whether a second alteration associated with cystic fibrosis (i.e., airway inflammation) could impact glucose control, animals were challenged with Aspergillus fumigatus. The A. fumigatus–sensitized CFTR−/− mice demonstrated similar fasting and stimulated glucose responses in comparison to nonsensitized animals. These studies suggest metabolic derangements in CFRD originate from an islet dysfunction inherent to the CFTR−/− state.
Cystic fibrosis is a systemic disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Some of the manifestations of this disease are caused by defective CFTR chloride channel function, but often they are related to cellular responses to mutant CFTR (1,2). In Caucasians, it is the leading life-limiting recessive genetic disorder (3). In 1938, the mean life expectancy for children with cystic fibrosis was <1 year (4). Over the past 30 years, the life expectancy of children with cystic fibrosis in the U.S. has increased from 16 to 32 years of age (4,5). The onset of diabetes heralds a great impediment to quality of life and is the leading comorbidity noted among the 23,000 patients with cystic fibrosis listed in the Cystic Fibrosis Foundation Patient Registry (5,6). The overall prevalence of cystic fibrosis–related diabetes (CFRD) has been estimated from 4.9 to 12% (5,6,7). In the cystic fibrosis population, the prevalence of CFRD appears to be increasing at a rate of 5% per year. By the age of 30 years, one study indicated 50% of cystic fibrosis patients had clinical diabetes (8). In addition, Hardin and Moran (6) report that the incidence of glucose abnormalities in adults with cystic fibrosis is 75%.
The prevailing mechanistic belief is that CFRD results from a combination of chronic pancreatitis and eventual loss of the islet cells, resulting in less insulin reserve and production, together with a variable state of insulin resistance (6,9–11). Clinical disease is associated with worsened glucose metabolism during times of lung exacerbation. Autopsy findings of individuals with cystic fibrosis have revealed minimal islet disease before the 2nd decade but extensive replacement of pancreatic acinar tissue with fibrous and fatty tissue. As patients age, autopsy data demonstrate the eventual loss of islet tissue and decreased β-cell numbers (12,13). Other findings include deposition of amyloid in the islets, a chronic architectural change, found in cystic fibrosis patients who also have CFRD (6).
As an alternative hypothesis, we question whether the origin of the glucose abnormality observed in CFRD is directly associated with CFTR mutation. The most common CFTR mutation (ΔF508) is a missense mutation that produces a misfolded version of CFTR; this mutation has been shown to trigger an endoplasmic reticulum overload response within cells (14), resulting in widespread changes in gene expression (1,15–17). Among the most prominent of these changes is an upregulation of proinflammatory cytokines associated with innate immunity (1). This may correlate with the observation that cystic fibrosis patients (18) and CFTR knockout mice (CFTR−/−) (19) show an exaggerated proinflammatory cytokine response to bacterial challenge compared with normal individuals. To address important questions relating to the formation of diabetes in cystic fibrosis, we developed a novel model involving administration of CFTR−/− mice with streptozotocin (STZ), an agent capable of inducing β-cell injury. These studies suggest that the CFTR mutation exacerbates islet cell dysfunction in CFRD after β-cell injury.
RESEARCH DESIGN AND METHODS
We used mice with the CFTR S489X−/− neo insertion, developed initially at the University of North Carolina (20). These mice were also modified with transgenic overexpression of gut-specific human CFTR, from the fatty acid binding–protein (FABP) promoter, to prevent intestinal obstruction and improve viability (21). These animals were developed on a background of C57BL/6J and FVB/NJ. Hence, age-matched C57BL/6J and FVB/NJ mice were used as control animals for all experiments. CFTR S489X−/− FABP-hCFTR+/+ (hereafter designated CFTR−/−), C57BL/6J, and FVB/NJ mice were housed under specific pathogen-free conditions at the University of Florida Animal Care Services according to National Institutes of Health guidelines and allowed food and water ad libitum. All experimental procedures were approved by the institutional animal care and use committee of the University of Florida.
Eight CFTR−/−, seven C57BL/6J, and six FVB/NJ mice received lactated ringers as a placebo via intraperitoneal injection at 7–9 weeks of age. Blood glucose levels were measured in CFTR−/−, C57BL/6J, and FVB/NJ mice at baseline. Random or fasting blood glucose values were checked weekly from the point of initial injection with lactated ringers until 4 weeks postinjection. In separate experiments, β-cell injury was induced according to the protocol by Ito et al. (22). Low-dose STZ (100 mg/kg; Sigma, St. Louis, MO) was given as a single intraperitoneal injection. CFTR−/− (n = 10), C57BL/6J (n = 11), and FVB/NJ (n = 6) mice received STZ at 7–9 weeks of age. Mice were monitored with blood glucose values determined weekly from the point of initial STZ injection until 4 weeks postinjection. STZ- and lactated ringer–administered mice underwent an intraperitoneal glucose tolerance test (IPGTT) 4 weeks after injection. Mice with random blood glucose levels >350 mg/dl at both week 1 and 2 after STZ administration were designated as early-onset diabetes. These mice were discontinued from the protocol, secondary to the health of the mouse, but also because of the inability to test blood glucose in excess of 600 mg/dl at time of IPGTT. Mice with fasting blood glucose levels >240 mg/dl at the end of 4 weeks post–STZ administration were classified as late-onset diabetes and were included in the IPGTT.
To assess the impact of lung injury on glycemia, we used a protocol by Muller et al. (23) previously demonstrating an increased inflammatory response. CFTR S489X−/− animals (4–6 weeks old) were sensitized to Aspergillus fumigatus crude protein extract (Greer Laboratories, Lenoir, NC). Briefly, animals were administered intraperitoneal injections of 200 μg A. fumigatus dissolved in 100 μl PBS on days 0 and 14 (n = 4). Aerosol challenge was performed with 0.25% A. fumigatus for 20 min in a 30 × 30× 20 cm acrylic chamber, using a jet nebulizer (model LC-D; PARI Respiratory Equipment, Midlothian, VA) with an air (room air) flow of 6 l/min on days 28, 29, and 30 after the sensitization. Nonsensitized control mice (n = 5) received intraperitoneal injections with PBS alone and were challenged with A. fumigatus along with sensitized mice. At 48 h after lung challenge, IPGTTs were performed. Age-matched nonsensitized and unchallenged control animals (n = 6) were also compared with mice receiving inhalation challenge.
Mice were fasted for 4 h before receiving 2 g/kg dextrose (50% dextrose solution) as an intraperitoneal injection. Glucose tolerance was monitored via tail vein sampling at time 0 (just before 50% dextrose solution injection), 30, 60, 120, and 180 min. Glucose was measured with a LifeScan OneTouch Ultra glucometer (reported in milligrams per deciliter).
Histology and immunohistochemistry.
Pancreas, lung, duodenum, and liver specimens were obtained immediately after the mice were killed at the end of the IPGTT and fixed in 10% neutral buffered formalin (Fisher Scientific, Pittsburgh, PA). Tissues were processed into paraffin blocks and sections stained with hematoxylin and eosin for morphologic evaluation. The entire pancreatic specimen was visualized under 10× magnification, and all islets were counted and inspected for insulitis. Any evidence of insulitis was scored (24). Insulin immunohistochemistry was performed as previously described, using guinea pig anti-insulin antibodies (1:2000; Dako, Carpinteria, CA) and routine avidin-biotin-peroxidase detection (Vectastain ABC kit; Vector Labs, Burlingame, CA) with 3,3′-diaminobenzidine (DAB staining kit; Vector Labs) as chromagen (24). Amyloid deposition was evaluated by staining with alkaline Congo Red (PolyScientific, Bay Shore, NY).
Human CFTR detection in tissue.
To ensure the absence of human CFTR (hCFTR) expression by the pancreas, after the CFTR−/− mice were killed, pancreas, lung, and small intestine were collected. Using surgical tools sterilized and sprayed with RNase Away (Molecular BioProducts, San Diego, CA), tissue was removed and immediately frozen in liquid nitrogen. After homogenizing 100 mg of tissue, mRNA was extracted, using Oligotex Direct mRNA columns (Qiagen, Valencia, CA). For RT-PCR, a Qiagen One-Step RT-PCR kit was used. Briefly, 14 ng of mRNA were incubated at 50°C with reverse transcriptase for 30 min with gene-specific primers for hCFTR (hCFTR forward: 5′-aaacttctaatggtgatgaccag; reverse: 5′-agaaattcttgctcgttgac) or B-actin (B-actin1: 5′-agctgagagggaaatcgtgc; B-actin2: 5′-accagacagcactgtgttgg). This was followed by incubating the samples at 95°C for 15 min to inactivate the reverse transcriptase. The cDNA was then amplified with the same primers for 30 cycles by PCR. The no–reverse transcriptase controls were subjected to the same PCR conditions and primers but in the absence of reverse transcriptase.
Data were analyzed via one-way ANOVA, using Bonferroni corrections for multiple comparisons to evaluate individual differences in strain and treatments. Glucose tolerance was calculated, using the percent change from the baseline fasting blood glucose value, and compared as the total area under the curve (trapezoidal rule) followed by one-way ANOVA calculations and Bonferroni corrections as described above. Differences between groups were considered significant if the Bonferroni-corrected two-sided P value was <0.05. Correlations of the number of pancreatic islets with blood glucose values were calculated with a Pearson calculation. All statistical analyses were conducted using SAS 9.1.2 software (Cary, NC).
Detection of hCFTR expression.
Tissue obtained from the small intestine of the CFTR−/− mice demonstrated hCFTR mRNA, whereas the lung and pancreas sections did not (Fig. 1).
Glucose challenge in mice receiving lactated ringers.
No significant differences were observed in the fasting glucose levels of lactated ringer–administered CFTR−/− mice in comparison to the C57BL/6J and FVB/NJ background strains (time 0, P = NS) (Fig. 2A). In addition, responses to glucose stimulation were similar among these three strains after intraperitoneal injection of 50% dextrose solution (P = NS) (Fig. 2A). These studies did not suggest intrinsic differences in glucose regulation by the CFTR−/− mice.
Glucose challenge in STZ-administered mice.
Fasting blood glucose levels were higher in STZ-administered CFTR−/− mice compared with C57BL/6J and FVB/NJ background strains subjected to identical treatment (288.4 ± 97.4, 168.4 ± 35.9, and 188.0 ± 42.3 mg/dl for CFTR−/−, C57BL/6J, and FVB/NJ, respectively; P < 0.05) (Fig. 2B). These glucose values remained higher throughout the entire 3-h time period after glucose infusion, but the percent increase did not achieve significance in comparison to the other STZ-administered strains (Fig. 2B). These results suggest that in the face of minimally compromised β-cell function (in this situation, due to chemically induced injury), there is indeed an underlying defect in glucose metabolism associated with deficiencies in CFTR. A small subset of both CFTR−/− (1 of 10) and C57BL/6J (3 of 11) mice developed diabetes early after STZ injection and were classified as early-onset diabetes, and they were thus removed from the animal protocol (Table 1). All early-onset diabetic mice were male (weight 25–30 g) and had received more total STZ dosage (100 mg/kg) than the female counterparts (weight 20 g).
Glucose challenge in mice with lung inflammation.
Interestingly, no changes in glucose, either fasting or stimulated, were observed in A. fumigatus–administered mice in comparison to age-matched PBS-treated or untreated controls (Fig. 3). This suggests that acute lung inflammation does not play a dominant role in the predilection to diabetes in cystic fibrosis.
Pancreatic acinar and islet histology.
Both lactated ringer–and STZ-administered CFTR−/− mice demonstrated focal mild acinar degeneration with fibrosis and inflammatory infiltrates consisting primarily of mononuclear cells (Fig. 4). Saponification of pancreatic fat was also observed in one animal (Fig. 4A). Though not subject to direct quantitation, intensive histological examination suggested that these local changes in pathology were similar among both lactated ringer–and STZ-administered CFTR−/− mice. No C57BL/6J or FVB/NJ mice demonstrated evidence of pancreatitis.
Next, we compared the pancreatic islets to identify wherein variances in islet inflammation, insulin content, or number might provide partial explanation for the observed differences in glucose regulation between these strains. Insulitis was not observed in any strain, in either the absence or presence of STZ administration. To the question of insulin content, no qualitative differences were observed, as determined by immunohistochemical evaluation (data not shown). Likewise, no significant differences in islet number were observed in comparison to CFTR−/−, C57BL/6J, and FVB/NJ strains (Fig. 5A), in either the absence or presence of STZ administration (all P = NS). Islet amyloid deposition was not observed in any strain either with or without STZ administration (data not shown).
However, a strong negative correlation between the number of islets and fasting blood glucose measurements were noted in STZ-administered CFTR−/− mice (r = −0.73, P = 0.02) (Fig. 5B). The absence of such a correlation in the other strains of mice suggests that the CFTR deficiency exacerbates glucose tolerance in injured islets and that glucose regulation in CFTR−/− animals is dependent on the number of available islets.
The work presented here demonstrates differences in pancreatic inflammation between CFTR−/− and control mice and an exacerbation of abnormal glycemic control in CFTR−/− mice after a low-grade chemical injury with STZ. Without STZ, CFTR−/− mice demonstrate focal inflammatory cell infiltrates and changes indicative of injury to the exocrine pancreas at baseline. This suggests the presence of intrinsic pancreatic disease leading to reduced effectiveness of β-cell function in cystic fibrosis (Fig. 6) and a need for adequate β-cell mass to maintain glycemic control.
The demonstration of higher fasting blood glucose levels and exaggerated IPGTT levels observed in STZ-administered CFTR−/− mice mimic key features of diabetes in cystic fibrosis. The response to islet injury is also suggestive of an inherent difference in function of CFTR-deficient β-cells. The absence of visible insulitis reinforces this conclusion. Although the background strains of the C57BL/6J and FVB/NJ mice maintained glucose control after STZ administration, despite a decrease in islets, the CFTR−/− mice were unable to maintain glucose control, and the degree of hyperglycemia was reflected by the decrease in islet number. The lack of lung disease and other clinical manifestations allow for the assessment of isolated influences. Taken collectively, these studies support the notion that CFTR−/− mice subjected to STZ administration provide an unequaled ability to recreate the CFRD clinical model.
Interestingly, the induction of airway inflammation in the CFTR−/− mice did not result in a difference in glycemic control, even though proinflammatory cytokine and IgE responses are greater in these mice than in control mice when exposed to A. fumigatus (23). This suggests that pulmonary inflammation, and subsequent insulin resistance, is not necessary for the predisposition to diabetes in cystic fibrosis, once again pointing to the role of intrinsic, CFTR-dependent abnormalities in the pancreas. Further studies evaluating the effect of chronic (versus acute) inflammation are needed. The impact of diabetes on lung inflammation can be further studied in this model as well.
C57BL/6J Cftr−/− mice, a variation of CFTR−/− mice, are the result of backcrossing CFTR−/− mice into a C57BL/6J strain. This breeding effectively causes an inability to wean to a solid diet, which had been previously afforded by the gut expression of FABP-hCFTR. This variation has previously been described to have intestinal inflammatory changes, including focal pancreatic acinar damage (25–27). In addition, these mice develop more of the cystic fibrosis phenotype, including lung pathology (27). Interestingly, the CFTR S489X−/− FABP-hCFTR+/+ model used in this experiment has not been previously reported to have pancreatic disease. However, in our series of experiments using this transgenic model, we found evidence of focal pancreatitis and saponification not previously described. This evidence of pancreatitis could impact β-cell function. Similarly, the unique correlation of islet numbers to fasting blood glucose may imply a dysfunction of CFTR−/− islets as well. Our conclusion is that CFTR−/− islets maintain glucose control by quantity of islets. In humans, proliferation of existing islets can be seen during times of insulin resistance or increased need (i.e., obesity or pregnancy); however, preliminary data by our laboratory has not revealed increased insulin resistance in the baseline CFTR−/− mouse as measured by fasting insulin levels (M.S.S., unpublished observations).
These findings provide the foundation for a large number of future studies aimed at addressing the pathogenesis of CFRD. Among them, our studies used mice 7–9 weeks of age in an attempt to age-match additional studies of type 1 diabetes in the NOD mouse model. However, future studies are required to address the relationship of age to the degree of glucose metabolism in the non–STZ-administered CFTR−/− model. This issue finds importance because age remains the most outstanding predictor of diabetes in the cystic fibrosis population.
As the population of individuals with CFRD grows, the need for animal models to study human disease becomes greater. The diagnosis of CFRD carries a poor prognosis, despite the lack of understanding regarding the influence on both lung and overall health. Through a better understanding of the etiology of CFRD, the full impact of disease can be seen, and strategies for a cure can be developed. The mouse model of this report provides one interesting opportunity to study the pathophysiology of CFRD and its systemic impact.
|Mouse strain .||n .||Early-onset diabetes .||Late-onset diabetes .||Total diabetes .|
|Mouse strain .||n .||Early-onset diabetes .||Late-onset diabetes .||Total diabetes .|
Mice were monitored weekly via random blood glucose levels immediately prior to and after injection with low-dose STZ. On the 4th week after STZ administration, 4-h fasting blood glucose was obtained prior to injection with 50% dextrose solution. Early-onset diabetes was defined as two consecutive blood glucose levels >350 mg/dl at week 1 and two post–STZ administration. Those mice were removed from the protocol and did not undergo a glucose challenge. Late-onset diabetes was defined as 4-h fasting blood glucose >240 mg/dl at the end of the 4-week period. All early-onset diabetic mice were males weighing 25–30 g, and thus they received the largest dosages of STZ administration. This effect was felt to be a dose effect of the STZ administration. In the STZ-administered CFTR−/− group, there were five males and five females. The male CFTR−/− mice weights were ∼25 g, whereas the C57BL/6J mice weighed 25–30 g. Female mice of both strains weighed ∼20 g. In the CFTR−/− group, four of five CFTR−/− males developed fasting hyperglycemia, and two of five females developed fasting hyperglycemia.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These studies were supported in part by a Lawson Wilkins Pediatric Endocrinology Society Clinical fellowship, a Cystic Fibrosis Foundation clinical fellowship, and National Institutes of Health Grants M01RR00082 and P01HL51811.
We thank Robert F. Schwartz, Stacy Binns, and Todd Brusko for their technical support to these studies.